Goat α mouse igg alexa657

Antibodies used for immunoblotting are as follows: Rat α-FLAG L5 (#637303; BioLegend), Rabbit α-HA, Rabbit α-GFP, Rabbit α-Sec62, Rabbit α-Sec63, Rabbit α-Sec61β, Rabbit α-BAG6, and Rabbit α-Sec61α (Chitwood et al., 2018 (link); Mariappan et al., 2010 (link); Snapp et al., 2004 (link)) are a gift from Dr. Ramanujan Hegde (MRC Laboratory of Molecular Biology, Cambridge, UK). Rabbit α-BiP (#11587-1-AP; Proteintech), Mouse α-HA (#901513; Biolegend), Mouse α-PDI (#MA3-018; Affinity Bioreagents), Goat α-Rat-HRP (#7077; Cell Signaling), Goat α-Mouse-HRP (#115-035-003; Jackson ImmunoResearch), Goat α-Rabbit-HRP (#111-035-003; Jackson ImmunoResearch), Goat anti-rat HRP (#7077S; Cell signaling), Goat α-RAT IgG-Cy2 (#112-225-167; Jackson ImmunoResearch), Goat α-Mouse IgG-Alexa657 (#A-21235; Invitrogen). Beads were purchased as follows: Strep-Tactin XT beads (#2-4010-010; IBA), Rat anti-FLAG L5 affinity gel (#651503; Biolegend), Protein A agarose (#CA-PRI-0100; Repligen), Mouse anti-HA magnetic beads (#88837; Pierce), Poly L-lysine (#OKK-3056; Peptides International). Rabbit Reticulocyte Lysate was purchased from Green Hectares (Ph:1-800-GHLYSAT). Detergents were purchased as follows: digitonin (EMD Millipore), Triton X-100 (Thermo Fisher Scientific), sodium deoxycholate (Sigma-Aldrich), SDS (Sigma-Aldrich), and Tween 20 (American Bioanalytical).

BiP-3xFLAG HEK293 cells (0.12 × 106) were plated on 12-mm round glass coverslips (Thermo Fisher Scientific) coated with 0.15 mg/ml poly-lysine in 24-well plates. The following day, cells were fixed and immunostained using a standard procedure (Sundaram et al., 2017 (link)). Rat anti-FLAG antibody (#637303; Biolegend) and mouse α-PDI (#MA3-018; Affinity Bioreagents) were used as primary antibodies. Goat α-RAT IgG-Cy2 (#112-225-167; Jackson ImmunoResearch) and Goat α-Mouse IgG-Alexa657 (#A-21235; Invitrogen) were used as secondary antibodies. Cells were imaged on Leica scanning confocal consisting of an inverted microscope (Leica SP6/SP8) and an HC PL APO 63×/1.4 (CS2 No: 11506350) oil objective lens (Leica) and was controlled by the Leica Application Suite X software. Sequential image scanning at 1× zoom, 100 Hz, 1,024 × 1,024 pixels, and with line averaging set at four was used to collect images for displayed images.